I. The Vi Antigen
The Vi antigen, a capsular polysaccharide, was first described by Felix et al, Lancet, 227:186-191 (1934). This capsular polysaccharide is present in Salmonella, such as S. typhi, S. paratyphi C, and S. dublin, as well as in Citrobacter freundii. Structurally, the Vi antigen is a linear polymer of .beta.-4,2-deoxy-2N-acetylgalacturonic acid with variable O-acetylation (Daniels et al, Infect. Immun., 57:3159-3164 (1989)). Its presence in S. typhi has been correlated, in vitro, with a significant decrease in lysis by serum, complement activation and phagocytosis (Looney et al, J. Lab. Clin. Med., 108:506-516 (1986)). Thus, the Vi antigen may act as a shield protecting S. typhi against the immune system.
A. The viaB Chromosomal Region and the Regulation of Expression of the Vi Antigen
Three widely separated chromosomal loci, viaA, viaB, and ompB are thought to be necessary for expression of the Vi antigen (Johnson et al, J. Bacteriol., 90:302-308 (1965); and Snellings et al, J. Bacteriol., 145:1010-1017 (1981)). Of these, the viaB locus is always found in Vi antigen-positive strains, and is thought to contain the genes encoding the enzymes necessary for the synthesis of Vi (Hashimoto et al, J. Bacteriol., 175:4456-4465 (1993); and Virlogeux et al, Microbiol., 141:3039-3047 (1995)). The viaB locus consists of 11 open reading frames (ORF) (FIG. 1A), of which the vipA and vipB genes encode the enzymes that synthesize the nucleotide sugar of the Vi polysaccharide, and the five vex genes (vexA-E) are thought to be responsible for translocation of the Vi antigen (Hashimoto et al (1993), supra). The first ORF of the viaB region, i.e., vipR (FIG. 1A), is a positive transcriptional regulator for its own expression, as well as for the expression of vipA, vipB, orf4, vipC, and perhaps others genes downstream of vipR (Hashimoto et al, J. Bacteriol., 178:1430-1436 (1996)). Furthermore, the promoter upstream of vipR also controls the transcription of (at least) vipA and vipB (encoding structural units of the Vi antigen), forming an operon within the viaB region. Another chromosomal region, the ompB operon, comprising the ompR-envZ genes, plays a role in the expression of the Vi antigen as a transcriptional regulator of viaB (Pickard et al, Infect. Immun., 62:3984-3993 (1994)). The ompR-envZ region forms part of the adaptive response of E. coli to conditions of high osmolarity. In S. typhi, the Vi antigen is osmotically regulated and ompR is necessary for its expression (Pickard et al, supra).
B. Relationship Between Exposure of the Vi Antigen and Immunoprotection
The Vi capsular polysaccharide of S. typhi is a virulence factor and a protective antigen in humans (Felix et al (1934), supra). Purified Vi polysaccharide is a licensed parenteral typhoid vaccine that elicits a moderate degree of protective immunity following inoculation with a single dose, and protection is mediated by serum IgG antibodies (Acharya et al, New England Journal of Medicine, 317:1101-1104 (1987); and Klugman et al, Lancet, 2:1165-1169 (1987)). In contrast, while attenuated S. typhi strain Ty21a, a licensed live oral vaccine, does not express the Vi antigen, nor does it elicit serum Vi antibody; nevertheless, Ty21a confers at least moderate levels of protection (Wahdan et al, J. Infect. Dis., 145:292-296 (1982); and Levine et al, Lancet, 1:1049-1052 (1987a)). It is believed that cell-mediated immune mechanisms mediate protection in this situation. Several new attenuated strains of S. typhi that express the Vi antigen in vitro have failed to elicit serum Vi antibodies when administered as oral vaccines, even though they elicit high titers of O and H antibodies (Tacket et al, J. Infect. Dis., 163:901-904 (1991); Tacket et al, Vaccine, 10:443-446 (1992a); and Tacket et al, Infect. Immun., 65:452-456 (1997)). The likely explanation for this phenomenon is that the expression of the Vi antigen is highly regulated in relation to osmotic stimuli (Pickard et al, supra). The supposition is that Vi antigen expression is interrupted, except when the bacteria are extracellular in the blood or other body fluids (e.g., bile).
It was postulated in the present invention that if Vi antigen expression is made constitutive, this would result in the stimulation of serum IgG Vi antibodies, thereby enhancing the overall protection against typhoid fever. It was also postulated in the present invention, that constitutive expression would improve the immune responses to foreign antigens expressed by attenuated Salmonella live vector vaccines and DNA-mediated vaccines.